We will continue the development of general and practical methods for the construction of mutants containing specific predetermined alterations within known nucleotide sequences. This will be done by: 1) introducing mutagenic nucleotides at specific points in the sequence using enzymatic techniques and 2) the incorporation of synthetic oligonucleotides, containing mutant sequences, into the DNA. We will develop and apply these techniques using the DNA of phage phi X174. We will use the DNA of phage phi X174, and various mutant strains to determine the sequence recognized by the E. coli K restriction and modification system. It has been reported that phi X174 DNA is transcribed in oocytes of Xenopus laevis. We will determine whether this transcription starts from a specific promoter, and if so map it.